Antimicrobial agents isolated from Aloe vera

ABSTRACT

Antimicrobial agents and method for isolation thereof from the gel liquid of  Aloe vera  includes at least one antimicrobial agent isolated from the clear gel isolated from the whole leaf of the  Aloe vera  plant, wherein the antimicrobial agent is an agent produced by the  Aloe vera  and/or indigenous bacteria that colonize the  Aloe vera  plant, is disclosed.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates in general to the field ofantimicrobial agents, and more particularly, to the characterization andisolation of agents that are responsible for antimicrobial activity ofAloe vera and its gel.

[0002] This application is a continuation of co-pending U.S. applicationSer. No. 09/262,731, filed Mar. 4, 1999, and claims the benefit under 35U.S.C. §119(e) of U.S. provisional application Ser. No. 06/077,145,filed Mar. 6, 1998.

BACKGROUND OF THE INVENTION

[0003] Without limiting the scope of the invention, its background isdescribed in connection with the identification of novel anti-microbialagents isolated from Aloe vera, as an example.

[0004] Heretofore, in this field, organisms that cause infectiousdisease, namely, viruses, bacteria, fungi and multicellular parasites,humankind has sought to control their morbidity and mortality. With theisolation and characterization of powerful antibiotics, beginning overhalf a century ago, the balance of power between humans and microbes hasbeen shifted toward humankind. For several decades after theintroduction of penicillin in the 1940's, for example, the conquest ofinfectious disease appeared imminent. The widespread use of antibiotics,added to the evolutionary flexibility of microbes, has made that victoryless than certain.

[0005] An increasing number of bacteria, fungi and other microbes aredeveloping resistance to antibiotics. A number of factors havecontributed to the increase in microbes that are resistant toantimicrobial agents. The use of combinations of anti-microbial agentsto treat nosocomial infections, particularly among patients whose immunesystems are compromised by AIDS, chemotherapy, or immunosuppressivedrugs, has led to a dramatic increase in multiple drug resistant (MDR)infections. Unfortunately, the future does not look bright in the waragainst infectious disease, as MDR strains of microbes continue toproceeding at an alarming rate. In fact, MDR strains are adapting fasterthan the introduction of new, more potent antibiotics.

[0006] Microbes have been developing strategies to cope with change forhundreds of millions of generations. In fact, some bacteria havegeneration cycles of 20 minutes, with each cycle providing theopportunity to evolve and adapt. Bacteria have adapted to anextraordinary range of conditions and developed defenses against allsorts of environmental threats, environmental and artificial. To amicrobe the human body is just another environment to colonize. Whileantibiotics are just another toxic environmental agent against which themicrobe must develop an escape strategy. For organisms with populationsthat have already adapted to such extreme environments as boilingunderwater hot-springs, learning to cope and evade antibiotics was onlya matter of time and evolution.

[0007] Overuse of antibiotics has contributed to the problem of MDRmicrobial strains. The indiscriminate use of antibiotics throughout theworld contributes to the continued emergence of MDR strains of bacteriasuch as Pseudomonas, Streptococcus and Staphylococcus. MDR strains haveevolved in large part because many patients fail to complete therequired course of antibiotic treatment, allowing stronger members ofthe microbial pool to be selected for in the next round of treatment.Increases in ear and sinus infections in children have been caused bythe use of antibiotics to treat viral infections, infections that arenot susceptible to antibiotic treatment. The current trend in medicineis to prescribe second-line and even last-resort antibiotics in place offirst-line antibiotics. Even when there is no reason to suspectresistance to first-line antibiotics, the drive toward using stronger,faster drugs is inevitable when faced with a sick patient. In the caseof recurrent lung infections in cystic fibrosis patients, physicianshave had no choice but to escalate to antibiotic treatment withsecond-line antibiotics, eventually causing the infecting bacteria tobecome resistant to all available antibiotics.

[0008] The emergence of MDR microbes has changed the balance betweenhost and parasite, from a position in which the medical community seemedpoised to achieve a conquest has lost ground in achieving a permanentconquest of microbial infection. But much has been learned in theprocess. Using a deeper understanding of microbes and their mechanismsof resistance, the biomedical community can continue to mount a broadarray of defenses against them. The microbes growing resistance totraditional antibiotics has renewed the attention to medical basics,such as public health measures, that include a renewed effort to steminfectious diseases by increasing hygiene. For example, in HIV-infectedpopulations, which have become breeding second-line and even last-resortantibiotics in place of first-line antibiotics. Even when there is noreason to suspect resistance to first-line antibiotics, the drive towardusing stronger, faster drugs is inevitable when faced with a sickpatient. In the case of recurrent lung infections in cystic fibrosispatients, physicians have had no choice but to escalate to antibiotictreatment with second-line antibiotics, eventually causing the infectingbacteria to become resistant to all available antibiotics.

[0009] The emergence of MDR microbes has changed the balance betweenhost and parasite, from a position in which the medical community seemedpoised to achieve a conquest has lost ground in achieving a permanentconquest of microbial infection. But much has been learned in theprocess. Using a deeper understanding of microbes and their mechanismsof resistance, the biomedical community can continue to mount a broadarray of defenses against them. The microbes growing resistance totraditional antibiotics has renewed the attention to medical basics,such as public health measures, that include a renewed effort to steminfectious diseases by increasing hygiene. For example, in HIV-infectedpopulations, which have become breeding grounds for resistant microbes,renewed educational outreach efforts focus on the use of prophylactics.

[0010] Nosocomial infections present the greatest threat toimmuno-compromised patients, because MDR microbes infect the mostvulnerable patients. It is the increase in MDR of microbes, and inparticular bacteria, that has led to a resurgence of interest inrevitalizing and improving basic techniques (like hand-washing) forpreventing the spread of infection. It has also increased the need foralternative, next-generation, anti-microbial agents. Theseanti-microbial agents, viz., anti-viral, anti-bacterial, anti-fungal andanti-parasitic, must also be safe for use in humans and other animals.

[0011] Antimicrobial-drug resistance is an increasingly important factorand poses a serious international challenge to public health incommunity and institutional settings. The list of resistant bacteria ofmajor public health importance includes those causing tuberculosis,gonorrhea, pneumococcal infections, and hospital-acquired enterococcaland staphylococcal infections. Antimicrobial-drug resistance hasresulted in prolonged and more serious illness, the use of moreexpensive and often more toxic drugs and drug combinations, andincreased fatality rates.

[0012] While pharmaceutical and biotechnology companies are constantlydeveloping novel products based on presently known antibiotics toovercome resistance, the next-generation of anti-microbial agents mustbreak from the known approaches to isolate and characterize theseactivities. A better understanding of the microbiology and moleculargenetics of microbial resistance is leading to the development of a newgeneration of anti-microbial agents that use an approach that isintended to attack standard mechanisms of action to kill bacteria orfungi. These so called new approaches to fighting microbial infectionsrely on variations of existing drugs having longer half-lives and morepotent effects, but rely on the existing database of pharmaceutics toattempt to outpace the microbes ability to evolve.

SUMMARY OF THE INVENTION

[0013] Both competition for nutrients and bacteriocin production play arole in determining the establishment of microbial communities innature. When analyzing symbiotic associations this may be furtherinfluenced by the presence of antimicrobial chemicals produced by thehost. In the case of Aloe vera barbedensis, the plant has been shown forcenturies to exert broad spectrum healing activities. The source of theantimicrobial agents isolated herein were determined, as were thedistinct populations of bacteria, and their dynamics within theindigenous microflora of Aloe vera. Localization of specific microbialpopulations was assessed using both direct culture of dissected plantmaterial and immunological detection within tissue sections. Relativesize and population diversity were determined through direct culture.Immunological detection demonstrated discrete populations withinspecific plant structures.

[0014] Bacterial identification was accomplished using standard stainingand biochemical analysis. To more completely identify the specificspecies of bacterium isolated and their role in the antibacterialactivity isolated herein, the environmental specimens were furtheranalyzed using restriction fragment length polymorphism (RFLP) analysis.RFLP analysis was used to differentiate the various species of Bacillusfound within the plant as well as definitive identification ofAeromicrobioum species and Curtobacterium species.

[0015] The efficacy of aloe liquid (see e.g., Coats, Aloe Vera: TheInside Story) as an antimicrobial agent is shown herein to have a widerange of gram negative and gram positive bacteria. The antimicrobialagents of the present invention are shown herein to effectively kill, orgreatly reduce or eliminate the growth rate of the following bacteria:Staphylococcus aureus, Streptococcus pneumonia, Streptococcus pyogenes,Pseudomonas aeruginosa, E. coli, Propionibacterium acne, Helicobacterpylori, and Salmonella typhi.

[0016] The anti-bacteriocidal activity demonstrated herein is not due tothe preservatives used in the preparation of the clear gel or theisolation of the antibacterial components, as the antimicrobial agentsisolated herein were isolatable from liquid collected directly fromfreshly cut whole leaves prior to used in the same killing assays.

[0017] In addition to the antimicrobial activities of its liquid, italso has been shown to be nontoxic even when taken internally. Theseproperties combined provide the impetus for the use of the antimicrobialagents isolated herein when used alone or in combination. The presentinvention demonstrates the isolation and identification of new,non-toxic, FDA approved antimicrobial agents that have been identifiedand isolated from the Aloe plant. These agents are efficacious andnontoxic, with a broad spectrum of antimicrobial properties.

[0018] Generally, and in one form of the invention, a compositionisolated from the gel liquid of Aloe vera including, at least oneantimicrobial agent isolated from the clear gel isolated from the wholeleaf of Aloe vera, wherein the antimicrobial agent is an agent producedby the Aloe vera or indigenous bacteria that colonize the Aloe vera, isdisclosed.

[0019] Furthermore, a method of decreasing the growth of a broadspectrum of bacteria including the steps of, isolating at least oneantibacterial agents from the clear gel of an aloe vera plant anddirectly contacting the bacteria with at least one antibacterial agentfrom aloe vera, is also disclosed.

[0020] The present invention is based on the recognition that aloe veraisolated have been used to treat, and increase the healing rate of,wounds and other infectious diseases. The present invention is alsobased on the recognition that antibacterial agents secreted by aloe veraand the bacteria that grow in the gel and rind of aloe vera and whichexhibit a wide range antimicrobial activity when exposed directly to thetarget microbe can be isolated. The antibacterial agents isolated hereinhave molecular weights of about: 555,000; 470,000; 240,000; 160,000;25,000 and 4,000 Daltons.

[0021] Also, the antibacterial agents isolated herein, are partiallysecreted by bacteria that grow in the gel and rind of Aloe vera. Thesecreted products of these species of bacteria exhibit a wide rangeantimicrobial activity when exposed directly to target microbes and eachother and include: Aerobacterium, Bacillus, Curtobacterium,Arthrobacter, Sporosarcina, and Clavibacter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] For a more complete understanding of the features and advantagesof the present invention, reference is now made to the detaileddescription of the invention along with the accompanying figure inwhich:

[0023]FIG. 1 is a taxonomic chart of the DNA fingerprint analysis ofbacterial isolates according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0024] While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts whichcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

[0025] Competition for nutrients and bacteriocin production play a rolein determining the establishment of microbial communities in nature.When analyzing symbiotic associations between plants and bacteria, thepresence of chemicals produced by the host as well as the bacteria playa role in the interaction. In the case of Aloe vera barbedensis, atropical or subtropical plant of the genus Aloe. These plants arecharacterized by having lance shaped leaves that contain a viscous cleargel. The leaves are given structural rigidity by hairlike connectivefibers. The clear gel of the aloe vera is to be distinguished from thethick, mucilaginous yellow juice that occurs about the base of the plantleaves and adjacent the rind of the leaf. This yellow juice, generallyknown as aloin, has been used for many years as an ingredient in manycathartics and purges.

[0026] The therapeutic qualities of the clear gel of aloe vera leavesdepend to a large extent on the freshness of the gel. For example, thegel of the Aloe plant has been used to stop the pain caused by a jellyfish sting. If the gel has been exposed to air and light for about oneand a half hours, however, the ability of the gel to reduce theconcomitant immediate type hypersensitivity form of inflammation isgreatly reduced.

[0027] The Aloe plant has been shown for centuries to exert broadspectrum of disease curing activities, these activities have not beencharacterized at the molecular biologic level. With the renewed interestin the isolation and characterization of antimicrobial agents, thepresent inventors recognized that in addition to the ability to promotethe healing of animal cells, that Aloe gel may also containantimicrobial agents.

[0028] Aloe Plant Antimicrobial Agents

[0029] A screen for antimicrobial activity of the aloe liquid wasdeveloped by the present inventors to quantify and qualify theantimicrobial activity of the aloe liquid. A number of strains ofbacterial were tested. The following strains of Streptococci pneumoniaewere obtained from the American Type Culture Collection: ATCC 51936,ATCC 51937, ATCC 51938, and ATCC 51422. Each of these has beenimplicated in severe cases of otitis media, and are multi-drug resistant(MDR). Culture of these microorganisms was on Brain Heart Infusion media(BHI) supplemented with 5% sheep red blood cells (SRBC). Incubation wasunder increased CO₂ conditions to enhance growth.

[0030] The organisms were initially grown overnight to log phase.Dilutions were made to obtain cultures of approximately 10⁵ cells/ml.These bacteria were inoculated into the appropriate media to which 90%aloe liquid (Coates Aloe International as well as freshly isolated) wasadded. The present inventors recognized that the aloe liquid isnon-toxic to animals in pure form, be it topical or internal, therefore,studies can be conducted using pure aloe liquid isolated and sterile.Positive and negative controls were used to verify the grown and killingdata, as well as the sterility of the components used. Proper steriletechnique was used throughout these studies. Inoculations were conductedunder a sterile hood or using standard bacteriologic techniques. Thefinal concentration of cells in the aloe liquid cell culture, andcontrol cultures, was 10⁴ cells/ml. Aliquots of the culture were sampledat 0, 6, 12, and 24 hours and plated onto BHI with 5% SRBC, without aloeliquid, to determine the number of viable cells remaining in theculture. The following data were collected (all counts represent theaverage of triplicate platings): TIME STRAIN 0 Hrs 6 Hrs 12 Hrs 24 HrsATCC 51936 5 × 10⁴ 3 × 10⁴ 1 × 10² 0 ATCC 51937 6 × 10⁴ 4 × 10⁴ 1 × 10²0 ATCC 51938 5 × 10⁴ 4 × 10⁴ 8 × 10² 0 ATCC 51422 8 × 10⁴ 8 × 10⁴ 7 ×10² 0

[0031] In addition to broth culture assays, diffusion plates were alsoused to assess the efficacy of the aloe liquid. BHI plates supplementedwith 5% SRBC were swabbed with overnight cultures of one of the strainsso that a lawn of growth would result upon overnight incubation. A wellwas cut into the center of each plate and filled with 100% aloe liquid(Coats Aloe International). The plates were incubated overnight andzones of inhibition measured the following day. Each of the strains ofStreptococcus pneumoniae exhibited strong susceptibility to aloe liquidas evidences by rings of inhibition averaging 2 cm in diameter.

[0032] The results of the assays of antimicrobial activity on thebacterial strains Helicobacter pylori ATCC 49503 and Streptococcuspneumoniae ATCC 35088. The assays were run twice in triplicate and thepercentages of killing activity compared to control cultures under thesame conditions without aloe product added. All cultures contained 5%sheep red blood cells for survival of these bacterial strains and wereincubated in atmospheres enriched in CO₂. Helicobacter Streptococcuspylori pneumoniae % Aloe Product % Killing % Killing 90% 33% at 6 hrs10% at 6 hrs 100% at 12 hrs 80% at 12 hrs 100% 18 hrs 75% 71% at 24 hrs43% at 24 hrs 50% 49% at 24 hrs 12% at 24 hrs 25% 34% at 24 hrs 9% at 24hrs

[0033] Source of the Aloe Bacteriocidal Activity

[0034] In order to more closely identify and isolate the source ofantimicrobial activity produced and isolated from the Aloe plant, thepresent inventors have analyzed the population dynamics within theindigenous microflora of Aloe vera. Localization and characterization ofspecific microbial populations was assessed using both direct culture ofdissected plant material and immunological detection within tissuesections. Relative size and population diversity were determined throughdirect culture. In situ hybridization using specific immunologicaldetection techniques demonstrated that discrete populations of bacteriaare located within, and are segregate into, specific plant structures.

[0035] Most bacterial identification is accomplished using standardstaining and biochemical analysis. The identification of bacterial andother microbes in environmental specimens requires attention to the factthat many microorganisms that are closely related members of the samegenus or extremely unique organisms may be isolated. Isolation methodsare often inadequate providing only broad classification at best.Therefore, in an attempt to clarify the classification of indigenousbacteria from Aloe vera barbedensis, Restriction Fragment LengthPolymorphism (RFLP) analysis was used to identify variations in thestrains of bacteria isolated. RFLP analysis indicated that variousspecies of Bacillus are associated with the Aloe plant, and also led tothe definitive identification of Aeromicrobioum species andCurtobacterium species.

[0036] The efficacy of aloe liquid (prepared according to the Coatsprocess, U.S. Pat. No. 5,356,811, incorporated herein by reference) wasused as an antimicrobial agent. Aloe liquid was shown to haveantibacterial activity against a wide range of gram negative and grampositive bacteria. The aloe liquid produced by the Coats process, andisolated from whole leaf, was shown to effectively kill: Staphylococcusaureus, Streptococcus pneumonia, Streptococcus pyogenes, Pseudomonasaeruginosa, E. coli, Propionibacterium acne, Helicobacter pylori, andSalmonella typhi.

[0037] The bactericidal activity of the aloe liquid was not due to thepreservatives used in the preparation of this product, as liquid wascollected directly from freshly cut whole leaves used in the samekilling assays, demonstrated identical results. The inventors recognizedthat one advantage to the use of aloe liquid as an antimicrobial agentis that it is nontoxic even when taken internally. New antimicrobialagent were identified and isolated from the aloe liquid are efficaciousand nontoxic, with broad spectrum properties.

[0038] Aloe Bactericidal Activity is not From Previously IdentifiedComponents

[0039] Previously isolated and identified individual aloe products asantimicrobial products were studied to confirm that these compounds werenot responsible for the anti-bacterial activity identified herein.Purified extracts of several known Aloe components were provided by Dr.I. Danhoff, including: Albarin, Awbarin, Anthraquinone, Aloin A, Aloin A& B, Aloe emodin, and Yellow sap components were tested in like assays.When used in culture killing assays, only Aloin A and Aloe emodin showedany inhibition of bacterial growth, however, neither were comparable tothe percent killing achieved using liquid from the whole leaf.

[0040] Isolation of Novel Aloe Constituents as Antimicrobial Products

[0041] Fractionation of fresh aloe liquid from whole leaf was conductedusing standard column chromatography on Sephacryl 300. The resultingfractions were collected, peaks combined, the components precipitated toconcentrates assayed for antimicrobial activity. Fractions containingmolecular weights of approximately 550,000; 470,000; 240,000; 160,000;25,000; and 4,000 Daltons were found to have varying degrees ofbactericidal or bacteriostatic activity.

[0042] When these fractions were analyzed using gel electrophoresis,they were found to be mixtures of 1 to 3 components, and are thusconsidered to be heterogenous mixtures of the same compound whosemolecular weight might be affected by glycosylation or association withlipid moieties. Alternatively, the fractions may contain proteins thathave undergone varying forms of degradation or cleavage duringprocessing. One the other hand, these fractions might indicate thatseveral different components were isolated.

[0043] Chromatographic Separation

[0044] A chromatographic separation gel to be used in the procedures ofthe present invention is a three dimensional network which has a randomstructure. Molecular sieve gels comprise cross-linked polymers that donot bind or react with the material being analyzed or separated. For gelfiltration purposes, the gel material is generally uncharged. The spacewithin the gel is filled with liquid and the liquid phase constitutesthe majority of the gel volume. Materials commonly used in gelfiltration columns include dextran, agarose and polyacrylamide.

[0045] Dextran is a polysaccharide composed of glucose residues and iscommercially available under the name Sephadex (Pharmacia FineChemicals, Inc.). The beads are prepared with various degrees ofcross-linking in order to separate different sized molecules byproviding various pore sizes. Alkyl dextran is cross-linked withN,N′-methylenebisacrylamide to from Sephacryl-S300 which allows strongbeads to be made that fractionate in larger ranges than Sephadex canachieve.

[0046] Polyacrylamide may also be used as a gel filtration medium.Polyacrylamide is a polymer of cross-linked acrylamide prepared withN,N′-methylenebisacrylamide as the cross-linking agent. Polyacrylamideis available in a variety of pore sizes from Bio-Rad Laboratories (USA)to be used for separation of different size particles.

[0047] The separation gel material swell in water and in a few organicsolvents. Swelling is the process by which the pores become filled withliquid to be used as eluant. As the smaller molecules enter the pores,their progress through the gel is retarded relative to the largermolecules which do not enter the pores. This is the basis of theseparation. The beads are available in various degrees of fineness to beused in different applications. The coarser the bead, the faster theflow and the poorer the resolution. Superfine is to be used for maximumresolution, but the flow is very slow. Fine is used for preparative workin large columns which require a faster flow rate. The coarser gradesare for large preparations in which resolution is less important thantime, or for separation of molecules with a large difference inmolecular weights. For a discussion of gel chromatography, seeFreifelder, Physical Biochemistry, Second Edition, pages 238-246,incorporated herein by reference.

[0048] The most preferred methods of gel filtration for use in thepresent invention are those using dextran gels, such as Sephadex, andthose using dextran-polyacrylamide gels such as Sephacryl which are ableto separate molecules in the 180 to 500 kiloDalton range.

[0049] In addition studies, the fractions were compared singly and invarious combinations in the bacterial killing assay. The level ofkilling achieved with whole aloe liquid could only be achieved when allthe fractions were combined, indicating that the unconcentratedcomponents together provide for aloe's broad spectrum antibacterialactivity.

[0050] In order to more closely identify the components responsible forthe broad spectrum antimicrobial activity observed for aloe liquid, andhaving isolated bacteria indigenous to the aloe plant, the presentinventors recognized that the bacteria might be the source of some orall of the broad spectrum antimicrobial activity identified herein.

[0051] Eight distinct microorganisms were repeatedly been isolated fromthe rind and gel of the Aloe plant. Population studies have indicatedthe relative abundance and locale of each of these representatives.Since many antimicrobial substances are known to be produced by otherbacteria, the production of such bacteriocins from each aloe isolate wasanalyzed. Three bacteria produced secretable products that were capableof killing one or more of the following indicator organisms, including:Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, and Enterococcus(Streptococcus) faecalis.

[0052] Using gel electrophoresis, comparison of the secreted productsand whole aloe liquid, three bands appear to be shared. Those ofmolecular weight 470,000; 160,000; and 25,000. Using antibody stainingtechniques, two of the bacteria displaying antimicrobial activity werelocalized within the rind of the plant; one at the gel rind interface,the other just below the outer cuticle of the plant, and within theparenchymal tissue. The distinct locations of the bacteria wereidentified by the present inventors to be in prime locations for theproducts secreted by the bacteria to enter the veins of the plant andbecome constituents of the plant liquid along with those of plantorigin.

[0053] Contributions from these bacteriocins provide part of the answerfor the broad spectrum activity of the aloe liquid isolated from thewhole leaf. None of the bacteriocins, alone or in combination, werecapable of killing all of individually killing the microbes previouslyshown to be affected. The secreted products of the individual bacteriacontribute some of the components identified in the fractionationstudies, but not all.

[0054] Aloe Plant Defense Proteins Capable of Antimicrobial Activity

[0055] As the bacterial secretion products did not fully explain theplants full antimicrobial activity, mixed fractions, rather than cleansingle component fractions, were studied. Plant defense proteins appearto make up, or be responsible for, the last anti-bacterial components.

[0056] Most bacterial identification is accomplished using standardstaining and biochemical analysis. In the identification ofenvironmental specimens, where many microorganisms are closely relatedmembers of the same genus or extremely unique organisms, these methodsare often inadequate providing only broad classifications at best. Tofurther the classification of indigenous bacteria from Aloe verapreviously isolated, RFLP (Restriction Fragment Length Polymorphism)analysis was initiated. These results have been helpful indifferentiating the various species of Bacillus found within the plantas well as assisting in the identification of Aeromicrobium,Curtobacterium, and Clavibacter isolates.

[0057] Microbiologists have historically relied on cultural andbiochemical analysis for the identification of microorganisms. Whilethis provides adequate information for classification to the genus andsometimes species level, it lacks the sensitivity required to detectstrain variations in many groups of bacteria. Such identifications areof particular importance when dealing with environmental samples whereselection pressures favor subtle variations within species. Bothmorphological and biochemical characters are often strongly influencedby the environment, and thus special procedures are required todistinguish genotypes from phenotypic variations.

[0058] Using standard bacteriological procedures, a variety of bacteriawere isolated from two different Aloe vera plants. These isolates wereidentified to species level. Representatives of the genus Clavibacter,Curtobacterium, and Arthrobacter (established plant symbiotes) andmultiple members of the genus Bacillus comprised the majority of theindigenous flora. There are over 100 different species of Bacillusestablished in Bergey's Manual of Systemic Bacteriology (many with minorbiochemical variations). Clavibacter and Curtobacterium were originallyplaced in the genus Corynebacterium, but have been separated based onrRNA patterns. Because the members of these groups are biochemicallysimilar, DNA fingerprinting was used to provide more definitiveclassification information including DNA polymorphisms.

[0059] Materials and Methods

[0060] PLANTS. Aloe vera plants were obtained from two sources: CoatsAloe International (Mexico) and Calloway Nurseries (Texas). Additionalwork was also conducted on a plant from Irving, Tex. (original originunknown) with similar results. The plants were maintained in theHaggerty Science building on the University of Dallas campus. Naturallight exposure was obtained by placing the plants in a hallway floor toceiling window on the east side of the building. Temperatures weremaintained around 25° C. Plants were watered weekly or as needed,allowing the soil to dry in between.

[0061] MICROBIAL ISOLATION. Leaves were cut at the base near the stalkwith a sterile scalpel. They were they washed with soap (DialAntibacterial Soap) to remove surface contaminants, rinsed throughlywith sterile water followed by a rinse with 70% ethanol, and allowed toair dry under a sterile hood. Gloves were used through out the isolationprocess to prevent possible contamination from other sources.

[0062] The leaves were cut into strips and finely minced in preparationfor tissue homogenization. One gram of plant tissue was suspended in 2mls of sterile saline and homogenized (Dounce vessel). The resultantsuspension was plated using serial dilutions onto Tryptic Soy Agar.Plates were incubated at 37° C., and inspected for growth at 24 and 48hours. Pure cultures were established from the individual, distinctivecolonies which grew from the suspension.

[0063] MORPHOLOGICAL AND BIOCHEMICAL CHARACTERIZATION. Gram stains andacid fast stains were done on 18 hour cultures. The spore stains weredone on five day cultures. Standard microbiological media (Difco andBBL) and formulations thereof, were used to determine both morphologicaland biochemical characteristics. Enterotubes in place of individual tubemedia for some of the assays. Resulting data was used in theclassification of the various isolates using Bergey's Manual ofDeterminative Bacteriology.

[0064] ISOLATION OF DNA. Cultures were grown overnight at 37° C. in 30ml Tryptic Soy Broth. Cells were collected by centrifugation,resuspended and lysed in 0.2 M glucose with lysozyme (10 ug/ml) andsubsequently digested with proteinase K. Proteins, carbohydrates, andlipids were removed from the suspension by phenol:chloroform:IsoamylAlcohol extraction. DNA was precipitated by addition of cold 95% ethanolat −20° C. The recovered DNA was resuspended in sterile Tris-EDTA bufferand contaminating RNA removed by overnight digestion with RNAse. Thiswas followed by a second proteinase K digestion, phenol:chloroform:Isoamyl alcohol extraction and ethanol precipitation. Purified DNA wasquantified spectrophotometrically.

[0065] DNA FINGERPRINTING. Five to 10 ug of DNA was completely digestedwith restriction enzymes (Promega) under the recommended conditions.Restriction fragments were separated from each other by electrophoresison horizontal agarose gels using 1% in Tris:Acetate:EDTA buffer. HindIII digested phage lambda fragments were used as molecular weightmarkers. Digestion patterns were compared and used as additionalclassification information.

[0066] Morphological and Biochemical Characterization.

[0067] A total of 20 bacterial cultures isolated from two separate Aloevera plants. Each was assayed using standard biochemical media. Isolateswere tentatively placed in a specific genus according to Bergey's Manualof Determinative Bacteriology.

[0068] Of the ten isolates from each plant, five from each of the twoplants were classified as members of the genus Bacillus. These isolateswere gram positive spore forming rods which were catalase positive.Minor variations in sugar utilization and exoenzyme productiondemonstrated a wide variety of species present within the plants. Twoisolates which were catalase negative were also classed as members ofthe Bacillus genus due to other biochemical reactions. Catalase positivenon-spore forming irregular rods which were oxidase negative representedmembers of Curtobacterium. Those which were oxidase positive isolatesrepresented members of Clavibacter, Arthrobacter, or Aeromicrobium. Allbacteria in these groups exhibited pleomorphic morphology, some withdistinct rod-cocci cycle. Catalase negative nonendospore formers wereplaced in the genus Listeria. Only a few cocci were isolated; all weregram positive. One sporeformer was isolated from Plant 1 and was classedas Sporosarcina. The remaining cocci were catalase positivenonsporeformers and were placed in the genus Micrococcus.

[0069] DNA Fingerprinting Analysis.

[0070]FIG. 1 is a taxonomic analysis and chart that was developed usingDNA fingerprint analysis and biochemical testing of the isolates. Bothgram positive rods and gram positive cocci were identified using gramstaining followed by biochemical and DNA fingerprint analysis. Theisolates identified included members of the Bacillus, Arthrobacter,Curtobacterium, Clavibacter, Aeromicrobium, Sporosarcina and Micrococcusspecies. TABLE 3 BIOCHEMICAL CHARACTERISTICS ISOLATES 1AI 3A 3B 3E 4A 4B5A 5B 6Ai 6Ao Characteristics Gram Stain + + + + + + + + + + MorphologyR R R R C P R P P R Spore + + + − + − − − + + Catalase + + +Sl + + + + + + Oxidase + − − + − + + − + + Glucose + + + Sl + + − + + +Lactose Sl − Sl − Sl + + − + + Arabinose + − Sl − + + − − Sl +Adonitol + − Sl + − + + + + + Sorbitol − − + − − − − − Sl − Lysine − −Sl Sl − − + + + + Ornithine − − Sl Sl − − + + + + Indole − − − − − − − −− − Citrate + − + − + + + + + + Urea + + + + + + + + + + VP − + + − − −− − + + Nitrate + + + + + + − + + + Starch + + + − + + − − + + DNAse +− + + + + + − + + MSA − G FG − G G − G FG FG

[0071] Population Dynamics among Indigenous Microflora Isolated FromAloe Vera

[0072] Both competition for nutrients and bacteriocin production play arole in determining the establishment of microbial communities.Symbiotic associations may be influenced by the presence of plantdefense compounds produced by the host. In the case of Aloe vera, theplant has shown to secret many anthraquinones (produced by a largenumber of plants), which have been shown to effectively killmicroorganisms. Studies were conducted to determine the populationdynamics within the indigenous microflora of Aloe vera. The localizationof specific microbial populations was assessed using both direct cultureof dissected plant material and immunological detection within tissuesections. Relative size and population diversity were determined throughdirect culture while the immunological detection demonstrated discretepopulations within specific plant structures.

[0073] Isolation revealed ten different microorganisms indigenous to theAloe plant. These studies were concentrated on determining thepopulation dynamics of the ten isolates. Comparative numbers of themicroorganisms at different levels within the plant were firstdetermined using standard bacteriological plating techniques. Microbialinteractions were examined using spot plates to determine competitionand tissue printing to determine the exact location within the plate ofeach of the microorganisms.

[0074] MICROBIAL ISOLATION. Leaves were cut at the base near the stalkwith a sterile scalpel. They were they washed with soap (DialAntibacterial Soap) to remove surface contaminants, rinsed throughlywith sterile water followed by a rinse with 70% ethanol, and allowed toair dry under a sterile hood. Gloves were used through out the isolationprocess to prevent possible contamination from other sources.

[0075] The leaves were cut into three segments: top, middle, and bottom.Each of there were processed separately. The segments were cut intostrips then finely minced in preparation for tissue homogenization. Inaddition, the rind was separated from the internal ‘gel’ of the plantfor some strips in each segment and processed. One gram of plant tissuewas suspended in 2 mls of sterile saline and homogenized (Douncevessel). The resultant suspensions were plated using serial dilutionsonto Tryptic Soy Agar. Plates were incubated at 37° C., and inspectedfor growth at 24 and 48 hours.

[0076] POPULATION ENUMERATION. The number of similar colonies from eachof the suspension was counted and recorded.

[0077] SPOT PLATES. Overnight cultures of each of the isolates wereswabbed onto Tryptic Soy Agar (TSA) plates to produce a lawn of growth.The growth of each of these was challenged by spotting the othersymbiotes on top of the lawn and allowing the organisms to growovernight at 37° C. A clear area around the spot indicated exclusivecompetition between the two organisms. A zone of inhibition whichappeared to have been there initially but had been filled in at a latertime was interpreted as a static competition, where increasingpopulation levels over came the initial exclusion.

[0078] TISSUE PRINTING. Leaves from the plants were cut and cleaned asfor the homogenization procedure. They were then cut into stripsapproximately 5 mm in length. These were placed onto nitrocellulosefilters and pressure was applied to facilitate the transfer of bacteriafrom the plant tissue onto the filter. The filters were either usedimmediately or stored dry at −4° C.

[0079] ANTIBODY PREPARATION. Cultures of each of the microorganisms weregrown up overnight in 10 mls of Tryptic Soy Broth. The cells werepelleted, resuspended in 1 ml of saline, and heated at 70° C. for 1 hourto inactivate the cells. Further inactivation was achieved by additionof formaldehyde to the cells at a concentration of 0.6%. Inactivationwas checked by plating both aerobically and anaerobically for 48 hours.No growth indicated complete inactivation.

[0080] Lowry protein assays were done to determine the antigenconcentration. Two hundred micrograms (μg) of antigen was administeredto Sprague Dawley rats in the presence of complete Freund's adjuvant.The second administration of antigen was fourteen days later in thepresence of incomplete Freund's adjuvant. The rats were bled ten dayslater and the serum isolated. Antibodies were purified usinghydoxyapatite.

[0081] IMMUNOBLOTTING. The tissue blots were blocked with PhosphateBuffered Saline (PBS) containing skim milk. Primary antibody was appliedto the blot for 1 hour. Subsequent washings with PBS removed unboundantibody. Second antibody (anti-rat IgG-peroxidase) was applied for 1hour. Unbound second antibody was washed off and the peroxidasesubstrate 4-chloro-1-napthol was used to stain the blot. Areas ofintense blue-black indicated areas of microbial localization. TABLE 4POPULATION ENUMERATION MICROORGANISM (per 50 ug tissue) SECTION/SITE 1 23 4 5 6 7 8 9 10 Total Bottom-Whole 1 15 1.5 9 58 1 0 0 0 0 84.5Bottom-Gel 0 0 0 40 0 0 1 0 300 0 340 Bottom-Rind 2.5 19 1 4 61 0 0 1 00 87.5 Mid-Whole 2.5 21 1 2.5 0 3.5 1 0 0 1 1.5 Mid-Gel 0 0 0 41 0 0 0 0500 0 541 Mid-Rind 6 24 2 1 0 4 1 0 0 1 39 Top-Whole 1.5 13 2.5 0 35 3 120 0 1 77 Top-Gel 4 19 1.5 0 0 2.5 0 0 0 5 32 Top-Rind 1 25 2 1 25 15.51 21 0 1 95.5

[0082] TABLE 5 MICROBIAL COMPETITION SPOT ISOLATE LAWN 1A 3A 3B 3E 4A 4B5A 5B 6Ai 6Ao 1A + ++ + ++ ++ ++ − ++ − 3A − − − −/+ − −/+ − − −/+ 3B −− − − − − − − +/− 3E − − + ++ − ++ − ++ − 4A − − − − − − − − − 4B − − ++− ++ ++ − ++ − 5A − − − −/+ − − − − − 5B Not Available 6Ai − − − − − − −− − 6Ao − +/− ++ +/− ++ − ++ − ++

[0083] Representatives of all the isolates except Clavibacter were foundpresent within the rind. Clavibacter dominated the gel region. A fewBacillus species, Aeromicrobium and Curtobacterium species were found inlow numbers within the gel. The majority of the isolates were associatedwith or immediately surrounding the tubule containing rind (Table 4). Onthe surface this result is surprising since the rind is the site ofyellow sap, comprised primarily of anthraquinones. These compounds havebeen classified as plant defense compounds. They are also extremelyunstable. Not only are anthraquinones readily inactivated throughoxidation, it is conceivable that many of the Bacillus species presentwithin the rind secrete enzymes which might assist their inactivation.One of the characteristics of Bacillus species is the prolific secretionof a wide variety of exoenzymes.

[0084] Location in the rind/tubules may also be a protective measure bythose microorganisms sensitive to some bacteriocin produced byClavibacter and thus reflect competitive exclusion betweenendosymbiotes. A review of population numbers reveals that although thevariety of microbes is small within the gel fillet, the populationdensity of those found in the gel is at least five times more dense thaneven the most prominent member of the rind microenvironment (Table 4).Members of the rind population, specifically Aeromicrobium, do showinhibitory activity against the gel isolates (Table 5). These organismscould establish the barrier between the rind and the secretions of thegel allowing the various rind organisms to live undisturbed.

[0085] Bacteria are known to produce substances known as bacteriocinswhich are capable of killing closely related species, a process termedcompetitive exclusion. The data show that various members of theindigenous microflora do produce bacteriocins setting up regions ofexclusive regions within the plant.

[0086] Because of the possibility that these bacteriocins interferedwith the identification of certain microorganisms within the gel andrind, immunoblotting of tissue prints was chosen as a more specificmethod for localization of the plant's microflora. Only fourmicroorganisms have successfully been localized using this method. Theinability to develop antibodies may be due either to low antigenicproperties of the remaining microorganisms or to the low populationnumbers within the plant itself making it difficult to transfersufficient numbers required for immunoblots techniques.

[0087] Bacillus 1A shows distinctive localization within the innertubules of the rind while Bacillus 6Ai and 3B appear to inhabit the moreexterior regions, even outside of the plant itself in the skin. Theseresults place the sporeforming coccus Sporosarcina (4A) at the interfacebetween the rind and the gel and within the gel itself. These resultsconfirm the population enumeration studies.

[0088] A wide range of bacteria are adapted to various microenvironmentsat the soil and air interface and are important in nutrient uptake,frost damage, and biological control of plant pathogens. There are alsothose associated with the plant surfaces, both root and aerial. Thelocation of these organisms reflects their ecology. Physiologically somemay be extracellular, multiplying within intercellular spaces but notpenetrating plant cell walls or entering protoplasts. Others may be ableto penetrate the higher plant cell. These associations ultimately leadto symbiotic interactions: commensalistic, mutualistic, or in someinstances parasitic. Parasitic conditions, however, are considered to berare with respect to plant-bacteria associations. The internalenvironment of healthy plants is not conducive to bacterial growth ingeneral, however, the acquisition and development of cell wall degradingenzymes and toxins leading to disease. Alternatively, the secretion ofcompounds with the ability to inhibit plant defense compounds may assistin the establishment of certain symbioses.

[0089] Aqueous compositions (inocula) of the bacteriocins isolated fromthe Aloe plane as described herein, comprise an effective amount of theantimicrobial agent dissolved or dispersed in a pharmaceuticallyacceptable aqueous medium. As used herein the terms “contact”,“contacted”, and “contacting”, are used to describe the process by whichan effective amount of a pharmacological agent, e.g., pure or dilutealoe gel, comes is direct juxtaposition with the target cell. The phrase“pharmaceutically acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human, such as the gel isolated for Aloevera as described herein.

[0090] The preparation of an aqueous composition that contains a proteinor proteoglycan, such as the active components derived from the bacteriaand aloe plant, is well understood in the art. Typically, suchcompositions are prepared as injectables, either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection can also be prepared. The preparation can alsobe emulsified.

[0091] Proteoglycans, for example, can be formulated into a compositionin a neutral or salt form. Pharmaceutically acceptable salts, includethe acid addition salts (formed with the free amino groups of theprotein) and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like.

[0092] Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug releasecapsules and the like.

[0093] For parenteral administration in an aqueous solution, forexample, the aloe liquid or gel can be used directly without any toxiceffects to the animal. Alternatively, the aloe solution and theantimicrobial agents identified herein, can be dissolved or resuspended,in a suitable buffer, if necessary. Liquid diluents can first berendered isotonic with sufficient saline or glucose.

[0094] To kill a cell in accordance with the present invention, onewould generally contact the cell with pure or diluted aloe gel in acombined amount effective to kill the microbial cell. The term “in acombined amount effective to kill the cell” means that the amount ofbacteriocins are sufficient so that, when combined within the cell, thecell is induced to undergo apoptosis or another form of cell death.Although not required in all embodiments, the combined effective amountof the isolated aloe gel compounds will preferably be an amount thatinduces significantly more cell death than the use of either elementalone, and most preferably, the combined effective amount will be anamount that induces synergistic cell death in comparison to the effectsobserved using either element alone.

[0095] While these particular aqueous solutions are especially suitablefor intravenous, intramuscular, subcutaneous and intraperitonealadministration, the aloe solution of the present invention can beadministered directly at full concentration. In this connection, sterileaqueous procedures to produce aloe gel can be employed, as will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 mL of isotonic NaCl solutionand either added to 1000 mL of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

[0096] While this invention has been described in reference toillustrative embodiments, this description is not intended to beconstrued in a limiting sense. Various modifications and combinations ofthe illustrative embodiments, as well as other embodiments of theinvention, will be apparent to persons skilled in the art upon referenceto the description. It is therefore intended that the appended claimsencompass any such modifications or embodiments.

[0097] References

[0098] Coats, B. 1995 Aloe Vera: The Inside Story, Coats.

[0099] Harlow E and Lane D. 1988. Antibodies: A Laboratory Manual, ColdSpring Harbor Press.

[0100] Holt C A. 1992. Detection and localization of plant pathogens,Tissue Printing. Reid P D, et al, editors. Academic Press.

[0101] Reid PD. 1992. Physical Tissue Printing. In: Tissue Printing,Reid P D, et al, editors. Academic Press.

What is claimed is:
 1. A composition derived from a clear aloe veramixture which has less than 1 ppm aloin content and which has beenprepared from aloe vera gel, consisting essentially of a fractionobtained by gel filtration of the clear mixture, wherein the fractionhas an average molecular weight of either approximately 550,000 or470,000 or 160,000 or 25,000 or 4,000 Daltons and has bacteriocidal orbacteriostatic activity, the composition further comprising one or moreantimicrobial agents, wherein at least one of the antimicrobial agentsis an agent produced by the Aloe vera or indigenous bacteria thatcolonize the Aloe vera.
 2. The composition of claim 1 wherein thecomposition is sterile.
 3. The composition of claim 1 wherein at leastone antimicrobial agent is secreted by Curtobacterium.
 4. Thecomposition of claim 1 wherein at least one antimicrobial agent issecreted by Clavibacter.
 5. The composition of claim 1 wherein at leastone antimicrobial agent is secreted by Arthrobacter.
 6. The compositionof claim 1 wherein at least one antimicrobial agent is secreted bySporosarcina.
 7. The composition of claim 1 wherein at least oneantimicrobial agent is secreted by Micrococcus.
 8. The composition ofclaim 1 wherein at least one antimicrobial agent is secreted byAeromicrobium.
 9. The composition of claim 1 wherein at least oneantimicrobial agent is secreted by an oxidase and lactose positiveBacillus.
 10. The composition of claim 1 wherein at least oneantimicrobial agent is secreted by a oxidase and lactose negativeBacillus.
 11. The composition of claim 1 wherein at least oneantimicrobial agent is secreted by aloe vera as a plant defensemechanism against microbial infection.
 12. The composition of claim 1,further comprising at least one antimicrobial agent selected from afraction having an average molecular weight selected from the groupconsisting of approximately: 555,000; 470,000; 240,000; 160,000; 25,000and 4,000 Daltons.
 13. A method of decreasing the growth of a bacteriacomprising: preparing a composition derived from a clear aloe veramixture which has less than 1 ppm aloin content and which has beenprepared from aloe vera gel, consisting essentially of a fractionobtained by gel filtration of the clear mixture, wherein the fractionhas an average molecular weight of either approximately 550,000 or470,000 or 160,000 or 25,000 or 4,000 Daltons and has bacteriocidal orbacteriostatic activity, the composition further comprising one or moreantimicrobial agents, wherein at least one of the antimicrobial agentsis an agent produced by the Aloe vera or indigenous bacteria thatcolonize the Aloe vera; and contacting said bacteria with saidcomposition.
 14. The method of claim 13, wherein the composition issterile.
 15. The method of claim 13, wherein the composition furthercomprises at least one antimicrobial agent is secreted byCurtobacterium.
 16. The method of claim 13, wherein the compositionfurther comprises at least one antimicrobial agent is secreted byClavibacter.
 17. The method of claim 13, wherein the composition furthercomprises at least one antimicrobial agent is secreted by Arthrobacter.18. The method of claim 13, wherein the composition further comprises atleast one antimicrobial agent is secreted by Sporosarcina.
 19. Themethod of claim 13, wherein the composition further comprises at leastone antimicrobial agent is secreted by Micrococcus.
 20. The method ofclaim 13, wherein the composition further comprises at least oneantimicrobial agent is secreted by Aeromicrobium.
 21. The method ofclaim 13, wherein the composition further comprises at least oneantimicrobial agent is secreted by an oxidase and lactose positiveBacillus.
 22. The method of claim 13, wherein the composition furthercomprises at least one antimicrobial agent is secreted by a oxidase andlactose negative Bacillus.
 23. The method of claim 13, wherein thecomposition further comprises at least one antimicrobial agent issecreted by aloe vera as a plant defense mechanism against microbialinfection.
 24. The method of claim 13, wherein the composition furthercomprises at least one antimicrobial agent selected from a fractionhaving an average molecular weight selected from the group consisting ofapproximately: 555,000; 470,000; 240,000; 160,000; 25,000 and 4,000Daltons